Title page for ETD etd-0598-12052

Footprint Analysis of the Transcriptional
Control of Glycogen Phosphorylase 2
in Dictyostelium Discoideum

Degree

Master of Science

Department

Biology (Molecular and Cellular)

Advisory Committee

Advisor Name

Title

Rutherford, Charles L.

Committee Chair

Esen, Asim

Committee Member

Favis, Reyna

Committee Member

Stevens, Ann M.

Committee Member

Keywords

cell differentiation

Dictyostelium discoideum

DNase I footprint analysis

transcription

gene expression

AT-rich

looping

large footprints

replication protein A

Date of Defense

1997-12-12

Availability

unrestricted

Abstract

Glycogen phosphorylase 2 (gp-2) is a key enzyme during the development of Dictyostelium discoideum. The gp-2 enzyme breaks down glycogen into glucose monomers that are subsequently used to synthesize the terminal end products of cellular differentiation. This gene is an ideal candidate for studying the process of selective gene expression because its product figures so prominently in the development of this organism, implying a dependable control mechanism responsible for its developmentally regulated expression. I present in this thesis the identification of several putative cis-acting elements of gp-2 as revealed through footprint analysis. Due to the extreme AT-bias characteristic of Dictyostelium promoters, footprinting conditions required intensive optimization with respect to template, nonspecific competitor, source of protein extract and DNase I digestion. Using an endlabeled fragment containing seven repeated sequences (3 TA boxes [TAATTATA], 2 TAG boxes [TAAAAATGGT] and 2 C boxes [ACCCACT]), purified replication protein A and several developmental nuclear extracts were tested for DNA binding activity. Small footprints were observed on the TAG and C boxes of the promoter for both protein sources. However, using a more sensitive footprinting strategy involving multiple rounds of primer extension, larger footprints spanning the same promoter regions were detected. In both cases, the appearance of the footprints coincided with the documented transcriptional activity of the gene. It can be concluded from the data obtained that the TAG and C boxes are very likely cis-acting elements involved in the regulation of gp-2 expression.